The invention relates to an oral formulation of methylglyoxal and/or its imino acid conjugates for human use and methods for preparing the compositions. Particularly, the invention relates to compositions comprising methylglyoxal and more particularly, imino acid conjugates of methylglyoxal. The present invention also relates to formulations of methylglyoxal and imino acid conjugates of methylglyoxal that can be used for the treatment and suppression of malignant diseases including but not limited to the cancers of Colon, Prostate, Pancreas, Lung, Oral cavity, Glioblastoma, and Leukemia.
Alpha-ketoaldehydes are a series of chemicals derived from glyoxal (CHOxe2x80x94CHO), a dialdehyde with two carbon atoms. Methylglyoxal (2-oxopropanal) is the simplest alpha-ketoaldehyde that occurs naturally in small quantities inside normal cells. It is continually formed from several metabolic sources. Within a cell, alpha-ketoaldehydes function in regulating cell division [1]
Methylglyoxal (CH3COCHO), with three carbon atoms (C-3), is the first member of an aliphatic series where each consecutive member is extended by a CH2 unit (i.e., C-4, ethylglyoxal CH3xe2x80x94CH2xe2x80x94COxe2x80x94CHO; C-5, propylglyoxal CH3xe2x80x94CH2xe2x80x94CH2xe2x80x94COxe2x80x94CHO; etc.)
Methylglyoxal is a normal cellular metabolite with potential anticancer properties.
Szent-Gyorgyi and his collaborators in their pioneering work on the biological role of methylglyoxal had put forward strong evidences of in vitro inhibitory effect of methylglyoxal on Sarcoma-180 cells [2]. Egyud and Szent-Gyorgyi showed that when methylglyoxal was injected into mice along with sarcoma 180 cells, no tumor developed and mice remained completely healthy [3]. Apple and Greenberg similarly demonstrated that methylglyoxal significantly inhibited sarcoma tumor growth [4,5].
High concentrations of exogenous methylglyoxal exhibit selective inhibitory activity toward rapidly proliferating tumor cells versus non-proliferating normal cells in vitro. The molecular basis of methylglyoxal toxicity is not clearly understood, but may involve inhibition of DNA and protein synthesis [6,7]. Indeed, methylglyoxal is known to form adducts with nucleic acids. Additionally Methylglyoxal specifically inhibits mitochondrial respiration and glycolysis in a wide variety of malignant cells, whereas the respiration in normal cells remains uneffected by it under identical conditions [8]. This effect of methylglyoxal is mediated via an inactivation of the enzyme glyceraldehyde-3-phosphate dehydrogenase, a key glycolytic enzyme, reported to be structurally and functionally altered in malignant cells [9]. Methylglyoxal specifically inhibits electron flow through Complex 1 of the mitochondrial respiratory chain of and inactivates the enzyme glyceraldehyde 3 phosphate dehydrogenase, to mediate a critical reduction in the intracellular levels of ATP, leading to the inhibition of respiration in malignant cells [10].
By using a model malignant cell, the Ehrlich ascites carcinoma (EAC) cell developed in mice, Halder et al have further shown that methylglyoxal inhibits both mitochondrial respiration and glycolysis in this type of cells [11]. As a consequence of inhibition of both mitochondrial respiration and glycolysis ATP levels in these cells have been found to be critically reduced, rendering the cells non-viable.
Methylglyoxal is a very reactive and unstable molecule and tends to oxidise/polymerise on exposure to air. However, aqueous solutions of methylglyoxal at a concentration of 40% w/v or less are relatively stable. It is due to this reason that commercially available methylglyoxal is a 40% solution in water.
Much work has been carried out on the conjugates of methylglyoxal. U.S. Pat. No. 4,066,650 discloses the addition products between ketoaldehydes and secondary amines. The patent describes method of preparation of the addition products and the pharmaceutical compositions for the treatment of cancer. U.S. Pat. No. 4,238,500 describes the novel condensation products of xcex1-ketoaldehyde with enediol and their pharmaceutical uses as anticancer, antihypotensive and analgesic compounds. U.S. Pat. No. 5,147,652 describes the liposome encapsulated ketaoaldehydes and their pharmaceutical use for the treatment of cancer and other non-self cells. U.S. Pat. No. 5,849,783 describes the physically and chemically latentiated methylglyoxal or xcex1-ketoaldehydes for treating the non-self cells which includes cancer.
However, in order to study the effect of methylglyoxal in human patients suffering with cancer there is a need to develop a convenient formulation that is user friendly.
To date, there is no formulation reported for the administration of methylglyoxal for human use.
There is a need, therefore, for formulating methylglyoxal into pharmaceutically acceptable composition(s) which can be conveniently given to cancer patients.
An aspect the present invention is to convert aqueous solution of methylglyoxal to a form that can be administered to cancer patients without losing its efficacy.
Another aspect of the present invention is to convert aqueous solution of methylglyoxal to a stable, solid form that can be used to administer methylglyoxal to cancer patients without losing its efficacy.
Another aspect of the present invention to formulate the imino acid conjugates of methylglyoxal into pharmaceutically acceptable composition(s) which can be conveniently given to cancer patients.
The present invention aims to present a formulation of methylglyoxal which can be given orally to cancer patients.
The present invention also aims to present formulations of imino acid conjugates of methylglyoxal which can be given orally to cancer patients.
In the present invention imino acid refers only to pyroglutamic acid. Alkyl and aryl are defined below as substituent R1. The general formula of these conjugates and pharmaceutically acceptable salts can be described as shown in the following structure (I) 
where R1 is any C1-C12 straight or branched alkyl group benzyl, or phenyl;
where R2 is H, or COR3 where R3 is any C1-C6 straight or branched alkyl group, benzyl or phenyl.
The invention provides a process for the synthesis of alkyl/aryl ester of pyroglutamic acid, said process comprising:
(a) mixing of pyroglutamic acid, potassium hydrogensulphate and alcohol;
(b) heating the mixture in Microwave (MW) oven with intermittent stirring or heating the mixture to reflux with benzene as co-solvent with continuous stirring;
(c) monitoring the progress of the reaction by TLC;
(d) diluting the reaction mixture with ethyl acetate and filtering out the solid mass
(e) evaporating the solvent to yield a colourless oil:
(f) purifying by silica gel column chromatography.
The invention also provides a process for preparing a conjugate of methyl glyoxal with alkyl/aryl pyroglutamate, said process comprising:
(a) mixing of alkyl/aryl pyroglutamate, methyl glyoxal and optionally bentonite or clay in Dioxan:water:2:1;
(b) heating the mixture in MW oven with intermittent stirring or heating to reflux with continuous stirring;
(c) monitoring the reaction by TLC;
(d) diluting the mixture with MeOH and filtering;
(e) concentrating the filtrate in Rotovapor;
(f) removing the residual water as an azeotropic mixture with ethyl acetate;
(g) purifying by silica gel column chromatography.
The invention also provides a process for preparing acetate of the conjugate of methyl glyoxal-alkyl/aryl pyroglutamate, said process comprising:
(a) sonicating a mixture of conjugate of methyl glyoxal-alkyl/aryl pyroglutamate, acetic anhydride and bentonite or clay;
(b) monitoring the reaction by TLC;
(c) diluting with chloroform and filtering the mixture;
(d) concentrating the filtrate in vacuo to yield a crude oily substance;
(e) purifying by silica gel column chromatography.
Other methods known in the art can be used to prepare the imino acid conjugates of methylglyoxal of formula I.
Pharmaceutically salts of the imino acid conjugates include the but are not limited to the following: acetate, ascorbate, benzoate, citrate, oxalate, stearate, trifluroacetate, succinate, tartarate, lactate, fumarate, gluconate, glutamate, phosphate/diphosphate, and valerate. Other salts include Ca, Li, Mg, Na, and K salts, halides, salts of amino acids such as lysine or arginine; guanidine, ammonium, substituted ammonium salts or aluminium salts. The pharmaceutically acceptable salts of the imino acid conjugate may be prepared by methods known in the art.
The more preferred methylglyoxal conjugates (MGC) are listed as below: 
The present invention provides compositions and methods for the presentation of methylglyoxal and/or its imino acid conjugates in pharmaceutically acceptable form for oral administration to cancer patients. In the compositions of this invention methylglyoxal and/or its imino acid conjugates remain physically and chemically stable and can be administered in various dosage forms at a drug dose meant to be effective to exhibit clinically significant anticancer activity.
The methods of this invention comprise, consist of, or consist essentially of administering orally to the mammal a therapeutically effective formulation of methylglyoxal or imino acid conjugates of methylglyoxal. An effective dose of methylglyoxal or its conjugates or pharmaceutically acceptable salts therof ranges from 1 mg/Kg. B. Wt to 300 mg/Kg. B. Wt (preferably 10-100 mg)/Kg. B. Wt) of the mammal, with the dose dependent on the effects sought, the manner of administration, and the cancer being treated). In accordance with good clinical practice, it is preferred to administer the formulation at a dose that will produce anticancer effects without causing undue harmful side effects. The formulation may be administered either alone or as a mixture with other therapeutic agents such as 5-fluorouracil, methotrexate, etoposide, paclitaxel, taxotere, doxorubicin, daunarubicin, vincristine, vinblastine and other such known and established anticancer drugs.
Methylglyoxal and/or its imino acid conjugates can be administered orally to human cancer patients simply by diluting with purified water to form a solution.
Methylglyoxal and/or its conjugates can be administered orally to human cancer patients by incorporating in a flavoured/sweetened syrup base.
Solution of methylglyoxal and/or its conjugates can be adsorbed onto inert excipients like colloidal silica to convert into a solid form for ease of administration.
Methylglyoxal solution or solution of imino acid conjugates of methylglyoxal can be lyophilized with the incorporation of cryoprotective agents exemplified by but not limited to mannitol, lactose, sorbitol, trehalose etc.
The lyophilized or the adsorbed form of methylglyoxal and/or its conjugates is the most suitable and user friendly form that can be dispensed in a sachet, ampoule, vial, filled into hard gelatin capsules or into soft gelatin capsules or compressed into tablets with or without the addition of excipients.
All the above delivery systems may contain added auxiliary agents such as filler, diluents, preservatives, stabilizers etc.
The unit dosage ranges from 60 mg to 18 gm, more preferably 600mg to 6 gm.